Hemophilia A is an X-linked disorder in which clotting factor VIII (fVIII) mutations lead to a loss of fVIII expression and serious bleeding. While bleeding episodes can be treated with plasma-derived or recombinant fVIII, up to 30% of hemophilia patients produce antibodies (inhibitors) to therapeutic fVIII. Our overall goal is to develop novel approaches for the induction of tolerance that can be applied to the reversal of undesirable immune responses, such as inhibitor formation in hemophilia. We have utilized immunoglobulin (Ig) fusion proteins delivered to B lymphocytes via retroviral vectors for the induction of tolerance. This technology is based on the well-established tolerogenicity of IgG carriers, onto which we engineer multiple epitope-containing polypeptides in frame at the N-terminus. The multiple epitopes processed and expressed via B-cell antigen presentation lead to tolerance and clinical efficacy in multiple autoimmune models and hemophilia. For example, in murine hemophilia, we have prevented and reversed inhibitor formation via this protocol. Moreover, our data also suggest that regulatory CD4+CD25+ T cells (Tregs) are required in this process. To further define the role of Tregs in gene therapy, and to translate these studies to the clinic, we propose three interrelated Specific Aims. First, we will determine the role of FoxP3 expressing T regs in gene therapy for tolerance. We use FoxP3-GFP knock-in mice (crossed into fVIII-/-) and FoxP3-GFP x DO11.10 mice to follow the kinetics of induction of T-regs during gene therapy for fVIII and ovalbumin, respectively. We also will induce/expand Tregs and transfer GFP+ cells to naove recipients to establish their role in tolerance. Next, in Aim 2, we will modulate the responsiveness of non-human primate T cells using B-cell gene therapy as follows: T cells from tetanus toxoid- and fVIII-vaccinated cynomolgus monkeys will be cultured with B cells transduced with a TT-Ig or fVIII-Ig constructs in vitro as a test for tolerance induction. Such transduced B cells will also be used for in vivo tolerance induction. Validation of this approach in rhesus macaques will also be pursued. In Aim 3, we will determine the efficacy of B-cell expression of fVIII epitopes to induce tolerance to fVIII in human T cells. Thus, T cells and T-cell lines from identical twins with severe hemophilia and an adult with mild hemophilia will be cultured with autologous B cell lines transduced with fVIII domains to extend Aim 2 in vitro. The accomplishment of our aims will enable us to create vectors that can be used therapeutically for the prevention and reversal of inhibitor formation. Hemophilia A is a life-threatening blood clotting disorder caused by mutations in factor VIII, a clotting factor. The current therapy for this disorder is to administer functional factor VIII intravenously, but a significant number of patients produce antibodies to the treatment, making it ineffective. Our goal is to develop and translate a novel gene therapy protocol which reverses this antibody formation by inducing tolerance to therapeutic factor VIII.